Lee Rubin - US grants

An important stage in the development of the nervous system takes place when neuronal processes reach their target cells and form synapses. Pre- and most postsynaptic cells interact and become structured for efficient transfer of information across the synapse. Such structuring is typified by that of the neuromuscular junction in which nerve endings somehow dictate the precise positions of acetylcholine receptors (AChRs) and acetylcholinesterase in the postsynaptic region. The purpose of experiments suggested in this grant proposal is to understand, in molecular terms, how AChRs are localized. This will first involve identifying chemical factors used by neurons to mark the synaptic region. Related factors eventually become localized in the muscle fiber's basal lamina and provide an accessible starting source for their purification. Secondly, the formation of an AChR cluster involves cytoplasmic, possibly cytoskeletal, elements which can be identified by making use of the observation that cells transformed with Rous sarcoma virus (RSV) are unable to cluster AChRs. This suggests that an essential component of AChR clusters is phosphorylated and disrupted by the transforming factor of RSV, pp60src. Finally, AChR clusters become localized above myonuclei and Golgi apparatus following a decrease in the movement of cellular organelles. The manner in which this colocalization occurs, as well as its functional consequences, will be investigated. The proposed experiments should provide information crucial to understanding molecular events which occur during normal synapse formation and synaptic functioning. They should also contribute to an understanding of a variety of neuro-developmental disorders. Finally, these experiments are designed to study a discrete, readily observable aspect of cell function caused by viral transformation and could well contribute to a greater understanding of the transformation process.

DESCRIPTION (provided by applicant): The research in this proposal will apply genetic, cell biological and chemical screening approaches with the goal of identifying novel treatments for type 2 diabetes (T2D), thus addressing thematic areas 1 and 2 of this grants program. Due to a relative deficiency in pancreatic beta cells, T2D patients are unable to produce sufficient insulin to control their blood glucose levels. If the number of beta cells in T2D patients could be increased, their glycemic control could be significantly improved, thus delaying or preventing the devastating consequences of chronic hyperglycemia. It is well established that beta cells possess the capacity to dramatically increase their numbers by replication, suggesting the possibility of harnessing this replicative potential as a therapeutic avenue for T2D. However, genes that specifically control beta cell replication are largely unknown. Likewise, small molecules that can boost beta cell replication rates have not been described. To identify genes that control beta cell replication, we propose to use microarray analysis to compare the transcription profiles of actively replicating populations of beta cells to the profiles of quiescent populations. In this way, we will be able to identify candidate regulatory genes whose expression either positively or negatively correlates with replication status. In parallel, we will undertake a high-throughput small molecule screen on cultured pancreatic islets to identify compounds that can stimulate the rate of beta cell replication in vitro. Genes and compounds identified by these two analyses will then be tested in mouse models to determine their ability to regulate beta cell replication in vivo. Thus, the studies in this proposal are likely to lead to the identification of novel therapeutic targets and drug candidates for the treatment of diabetes. PUBLIC HEALTH RELEVANCE: The incidence of type 2 diabetes is rapidly increasing in the United States and worldwide. The negative impacts of long-term diabetes on a patient's health are significant, and carry with them a heavy financial burden. The therapies that will be developed as a result of the proposed research could be used to treat type 2 diabetes, and delay or prevent the devastating consequences of long-term disease. Thus, these therapies will both alleviate patient suffering while simultaneously reducing the financial costs of this disease.

Late onset neurodegenerative diseases, such as Alzheimer?s disease (AD), affect more than 7 million Americans, with the associated healthcare costs currently reaching hundreds of billions of dollars per year (and constantly rising). It is known that the pathology of AD involves many more cell types than the neurons of the hippocampus and cortex. The cells that comprise the brain vasculature, including the endothelial cells, pericytes, astrocytes and smooth muscle cells are critically important in maintaining the balance of health and disease in the brain. In particular, many properties of the endothelial cells, including their roles in establishing the blood-brain barrier (BBB), delivering nutrients to the brain, and regulating the proliferation of neural stem cells, are essential to proper brain function. Studies from our lab and others have demonstrated that brain vasculature can be restored even after it has been damaged, suggesting new strategies for treating neurodegenerative disorders via improving the integrity of brain vasculature. In experiments detailed in this application, we propose to both identify and correct processes within the cells of the brain vasculature that are known to be affected in Alzheimer?s disease and other dementias. Some of our work is based on the acknowledgement that aging is the major risk factor for dementia and is also characterized by declining vasculature. As a step toward obtaining a comprehensive understanding of aging-associated changes in the brain, our lab recently published a large single-cell RNAseq study comparing young and old mouse brains. Here, we propose to exploit our knowledge of the gene expression changes that define the aging process to identify cellular and molecular factors critical to brain blood vessel function and the maintenance of the BBB in a mouse model of AD. First, we will test several different hypotheses concerning the cellular and molecular bases for the vascular defects in the AD brain. Surprisingly, recent literature suggests that some of these changes are mediated by soluble factors and may be reversible. To explore this possibility in greater detail, we will use our knowledge of the CNS network of cell-cell interactions mediated by secreted factors to identify potentially correctable changes that occur in AD vasculature. Finally, we will use our lab?s expertise in human induced pluripotent stem cells (iPSCs) to employ an in vitro model of the BBB. This in vitro platform will serve as an important complementary approach to the in vivo mechanistic evaluation of putative aging or rejuvenation factors in human brain vascular cells. At the same time, we propose modifications of the current in vitro system that should improve its ability to recapitulate properties of the in vivo BBB. Together, our proposed studies seek to identify and validate new modulators of brain vasculature and to elucidate how the functions of these modulators play a role in the maintenance or degradation of the BBB in dementia.

Late onset neurodegenerative diseases together affect more than 7 million Americans with associated healthcare costs currently reach hundreds of billions of dollars per year. Cognitive decline is a common feature of many of these diseases, especially Alzheimer?s disease, Parkinson?s disease, and vascular dementia. In spite of exciting progress being made in studying those disorders, currently, there are no available therapeutics capable of improving cognition. Therefore, it came as a surprise when a set of observations from a few labs, including ours, supported the notion that recovery of brain function after damage to the CNS might be achievable. Much of the data was obtained from studies of heterochronic parabiotic mice ? young and old mice whose circulatory systems had been surgically joined. Our additional studies were equally exciting in that they demonstrated that injection of a single factor, GDF11, a normal serum protein, into aged mice was also able to improve important properties of the CNS. Specifically, GDF11 stimulated neurogenesis, increased neural activity and improved vascular structure. Surprisingly, we found that GDF11 does not cross the blood- brain barrier and instead may exert its effects by acting directly on aging brain vasculature. This proposal focuses on understanding in much greater detail how GDF11 exerts these ameliorative effects on the CNS. First, we will use a combination of histological, molecular and transcriptomic methods to investigate the effects of GDF11 on the cells of the brain more broadly. We will employ several measures including markers of neural activity, neurogenesis, angiogenesis, as well as changes in gene expression of the different cell types, and we will determine the sequence of GDF11?s actions (testing the hypothesis that GDF11?s neural effects are indirect and follow direct effects on brain vasculature). Next, we will compare GDF11?s effects on cells of the CNS with effects of other TGF?-family ligands such as GDF8, TGF?2 and modified forms of GDF11. Identifying the most effective ligand will help us understand the molecular changes these ligands produce, as well as position us to develop effective therapeutics in the future. Finally, our unpublished findings show that GDF11 and the components of its signaling pathway are expressed by multiple brain cell types well into adulthood. We will compare and contrast the functions of systemically injected GDF11 with those of GDF11 acting from within the brain. We will use a combination of histology and genetic perturbation to quantify the expression of GDF11 and its receptors across various regions the brain and how they are altered by aging. We will then measure the consequences of reducing brain GDF11 on neurogenesis and neural function. This will provide a better understanding of what might happen if systemic GDF11 gained direct access to neural cells in diseases in which the blood-brain barrier becomes compromised. From this work, we hope to gain a comprehensive understanding of the effects of GDF11, how they relate to those of other TGF?-family ligands, and what benefits to brain function may be achieved by administering these factors.